Oxazolidine, 3-butyl-2-(1-ethylpentyl)-

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2009-04-01 to 2009-04-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

adopted 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Version / remarks:

adopted 2002

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The concentration and stability of the test material in the test were verified by chemical analysis on Days 0, 5, 8, 12, 15 and 20 (fresh media) and Days 1, 6, 9, 13, 16 and 21 (old media).

Vehicle:

no

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Source: in-house laboratory cultures
- Age at study initiation: approximately 24 h

ACCLIMATISATION
- Type and amount of food: suspension of algae (Chlorella sp.)
- Feeding frequency: daily
Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water in a temperature controlled room at approximately 20 °C. The lighting cycle was controlled to give a 16 hours light (max. 600 lux) and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

no

Hardness:

144 to 104 (mg/L as CaCO3)

Test temperature:

20.0 - 21.0 °C

pH:

Control: 7.7 - 8.2
0.10 mg/L: 7.7 - 8.2
0.32 mg/L: 7.7 - 8.2
1.0 mg/L: 7.7 - 8.2
3.2 mg/L: 7.8 - 8.2
10 mg/L: 7.8 - 8.2

Dissolved oxygen:

Control: 95 - 110 %ASV
0.10 mg/L: 95 - 110 %ASV
0.32 mg/L: 93 - 110 %ASV
1.0 mg/L: 96 - 110 %ASV
3.2 mg/L: 94 - 110 %ASV
10 mg/L: 81 - 103 %ASV
ASV = Dissolved oxygen concentration expressed as a percentage of Air Saturation Value

Salinity:

not applicable

Nominal and measured concentrations:

Nominal concentrations: 0.10, 0.32, 1.0, 3.2, 10.0 mg/L
Due to the rapid hydrolysis of the test item, steady measured concentrations of the test item were not achievable. The hydrolysis products could not be detected by the analytical method due to possible but unconfirmed adsorption to surfaces.

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 mL glass flasks, covered with plastic lid
- Renewal rate of test solution: daily
- No. of organisms per vessel: 1
- No. of vessels per concentration: 10
- No. of vessels per control (replicates): 10
- Feeding: Each daphnid was fed with unicellular algal culture (Chlorella sp.), daily. Feeding was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day, dependent on the age and size of the animals. Equal amounts of food were given to each daphnid.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated tap water
- Total organic carbon: average 1.041 mg/L
- Total pesticides: below LOD
- Total chlorine: average 0.297 mg/L
- Sodium: 27.250 mg/L

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for 21 days
- Light intensity: 501 to 514 lux

EFFECT PARAMETERS MEASURED :
immobilisation, reproduction.

On a daily basis the numbers of live and dead of the "Parental" (P1) generation, the numbers of live and dead "Filial" (Fi) Daphnia and the number of discarded unhatched eggs were counted. An assessment was also made of the general condition and size of the parental Daphnia as compared with the controls.
The number of Daphnia with eggs or young in the brood pouch was determined daily. Young daphnids were considered to be dead if no sign of movement was apparent during microscopic examination. Adult Daphnia which were unable to swim for approximately 15 seconds after gentle agitation (ie. immobile), were considered to be dead. An immobilisation criterion for the young daphnids was considered to be inappropriate due to the large numbers of off-spring produced in the flasks.
At the end of the test, the length of each surviving parent animal was determined.

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

7.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: 95% confidence limits 3.5 - 56.4 mg/L

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

5.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: 95% confidence limits 2.3 - 26.1 mg/L

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

2.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: 95% confidence limits 1.5 - 5.7 mg/L

Duration:

14 d

Dose descriptor:

EC50

Effect conc.:

2.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: 95% confidence limits 1.5 - 5,7 mg/L

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

2.5

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: 95% confidence limits 0.97 - 4.3 mg/L

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

1.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% confidence limits 0.99 - 3.2 mg/L

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

Due to the rapid hydrolysis of the test item, steady measured concentrations of the test item were not achievable. The hydrolysis products could not be detected by the analytical method due to possible but unconfirmed adsorption to surfaces.

Lethal Effects on the Parental Generation (P1)
Mortality (immobilisation) occurred at the highest test concentration of 10 mg/L resulting in 100 % mortality by Day 5. Significant mortality (immobilisation) also occurred throughout the test in the 3.2 mg/L test group resulting in 60 % mortality by Day 21 indicating a prolonged toxic effect attributable to exposure of Daphnia magna to the test material. Mortality was also observed at the test concentrations of 0.32 mg/L on Day 4 and 1.0 mg/L on day 2. However, statistical analysis of the mortality data using the corrected chi-squared statistic showed that the observed mortalities in the 0.32 and 1.0 mg/L test groups were not significantly different (P > 0.05) when compared to the control group. No mortalities occurred at the 0.10 mg/L test concentration throughout the test.

Sub-lethal Effects on the Parental Generation (P1)
There was a significant effect on size and colour of the daphnids in that 100 % of the surviving daphnids on Day 4 at the test concentration of 10 mg/L were markedly smaller and paler in colour than the control animals. There was a significant effect on colour of the daphnids in that 100 % of the surviving daphnids at the test concentration of 3.2 mg/L were markedly paler in colour than the control animals. The daphnids at the remaining test concentrations were observed to be the same size and colour as the control animals. After 21 days there were no statistically significant differences (P ≥ 0.05) between the control and the 0.10 and 0.32 mg/L test groups in terms of the number of live young produced per adult. The 1.0 and 3.2 mg/L test groups showed a statistically significant difference from the control and the remaining test groups after 21 days in terms of producing fewer numbers of live young per adult. The 10 mg/L test group data was not included in the statistical analysis as exposure to the test material eliminated all the daphnids prior to Day 21 of the test.
After 21 days the length of each surviving adult was determined. The results showed that there were no statistically significant differences (P ≥ 0.05) between the control and the 0.10 and 0.32 mg/L test groups in terms of length of the daphnids after 21 days exposure to the test material. The 1.0 and 3.2 mg/L test groups showed a statistically significant difference from the control and the remaining test groups after 21 days in terms of length of the daphnids. The 10 mg/L test group data was not included in this analysis as exposure to the test material eliminated all the daphnids prior to Day 21 of the test.

Effects on the Filial Generation (Fi)
Information on the effects of the test material on the Fi generation is limited, since, by study design, the young are removed soon after liberation from the brood pouch. However, an assessment made at each media renewal showed the "filial" daphnids produced by all the test groups were in the same general condition as the young produced by the controls over the duration of the test. Young were first produced in the control test group on Day 8 of the test. Due to the toxic effect of the test material the parental generation (Pi) of the 10 mg/L test group were eliminated prior to the production of young. Numbers of unhatched eggs and dead young were low in all control and treatment groups surviving to maturation.

Validity criteria fulfilled:

yes

Conclusions:

The long-term and reproduction toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and the following results, based on nominal test concentrations, were achieved: The 14- and 21-Day EC50 (immobilisation) values for the parental Daphnia generation (P1) were 2.8 and 2.5 mg/L, respectively.
The 21-Day EC50 (reproduction) was 1.8 mg/L.
The "Lowest Observed Effect Concentration" (LOEC) and the "No Observed Effect Concentration" (NOEC) were 1.0 and 0.32 mg/L, respectively.

Executive summary:

A study was performed to assess the long-term and reproduction toxicity of the test material to Daphnia magna, according to OECD Guideline 211 and EU method C.20. Based on the results of an acute toxicity test to Daphnia magna (see IUCLID Section 6.1.3 Short-term toxicity to aquatic invertebrates), the following test concentrations were used for a total exposure duration of 21 days: 0.10, 0.32, 1.0, 3.2 and 10 mg/L.

For each concentration a single daphnid was placed in 100 mL of the test preparation in 150 mL glass flasks which were then covered with a plastic lid to reduce evaporation. For each test and control group ten replicate test vessels were prepared. The test preparations were renewed daily. The control group was maintained under identical conditions but not exposed to the test material. On a daily basis the numbers of live and dead of the "Parental" (Pi) generation, the numbers of live and dead "Filial" (Fi) Daphnia and the number of discarded unhatched eggs were counted. An assessment was also made of the general condition and size of the parental Daphnia as compared with the controls. The number of Daphnia with eggs or young in the brood pouch was determined daily. Young daphnids were considered to be dead if no sign of movement was apparent during microscopic examination. Adult Daphnia which were unable to swim for approximately 15 seconds after gentle agitation (ie. immobile), were considered to be dead.

Mortality (immobilisation) occurred at the highest test concentration of 10 mg/L resulting in 100 % mortality by Day 5. Significant mortality (immobilisation) also occurred throughout the test in the 3.2 mg/L test group resulting in 60 % mortality by Day 21 indicating a prolonged toxic effect attributable to exposure of Daphnia magna to the test material. Mortality was also observed at the test concentrations of 0.32 mg/L on Day 4 and 1.0 on day 2 but was not considered significant following statistical analysis. No mortalities occurred at the 0.10 mg/L test concentration throughout the test.

Offspring daphnids produced by all the test groups were in the same general condition as the young produced by the controls over the duration of the test. Young were first produced in the control test group on Day 8 of the test. Due to the toxic effect of the test material the parental generation (Pi) of the 10 mg/L test group were eliminated prior to the production of young. Numbers of unhatched eggs and dead young were low in all control and treatment groups surviving to maturation.

 

Based on these observations, the following results were calculated: The 14 and 21 -Day EC50 (immobilisation) values, based on nominal test concentrations, for the parental Daphnia generation (P1) were calculated to be 2.8 and 2.5 mg/L with 95 % confidence limits of 1.5 - 5.7 mg/L and 0.97 - 4.3 mg/L, respectively. The 21-Day EC50 (reproduction) based on nominal test concentrations was 1.8 mg/L with 95 % confidence limits of 0.99 - 3.2 mg/L. The "Lowest Observed Effect Concentration" (LOEC) and the "No Observed Effect Concentration" (NOEC) based on nominal test concentrations were 1.0 and 0.32 mg/L, respectively.

Due to the rapid hydrolysis of the test item, steady measured concentrations of the test item were not achievable. The hydrolysis products could not be detected by the analytical method due to possible but unconfirmed adsorption to surfaces.

Description of key information

The long-term and reproduction toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and following results, based on nominal test concentrations were achieved:

The 14 and 21-day EC50 (immobilisation) values for the parental Daphnia generation (P1) were 2.8 and 2.5 mg/L.

The 21-day EC50 (reproduction) was 1.8 mg/L.

The "Lowest Observed Effect Concentration" (LOEC) and the "No Observed Effect Concentration" (NOEC) were 1.0 and 0.32 mg/L, respectively.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.32 mg/L

Additional information

A study was performed to assess the long-term and reproduction toxicity of the test material to Daphnia magna, according to OECD Guideline 211 and EU method C.20. Based on the results of an acute toxicity test to Daphnia magna (see IUCLID Section 6.1.3 "Short-term toxicity to aquatic invertebrates"), the following test concentrations were used for a total exposure duration of 21 days: 0.10, 0.32, 1.0, 3.2 and 10 mg/L.

Due to the rapid hydrolysis of the test item, steady measured concentrations of the test item were not achievable. The hydrolysis products could not be detected by the analytical method due to possible but unconfirmed adsorption to surfaces. Based on the observations during the study, following results were calculated: The 14 and 21-day EC50 (immobilisation) values, based on nominal test concentrations, for the parental Daphnia generation (P1) were calculated to be 2.8 and 2.5 mg/L with 95 % confidence limits of 1.5 - 5.7 mg/L and 0.97 - 4.3 mg/L, respectively. The 21-day EC50 (reproduction) based on nominal test concentrations was 1.8 mg/L with 95 % confidence limits of 0.99 - 3.2 mg/L. The "Lowest Observed Effect Concentration" (LOEC) and the "No Observed Effect Concentration" (NOEC) based on nominal test concentrations were 1.0 and 0.32 mg/L, respectively.